Int.J.Curr.Microbiol.App.Sci (2017) 6(12): 275-297

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 12 (2017) pp. 275-297 Journal homepage: http://www.ijcmas.com

Original Research Article https://doi.org/10.20546/ijcmas.2017.612.034

A Study on the Diversity and Habitat Specificity of Macrofungi of Assam, India

Assma Parveen, Lipika Khataniar, Gunajit Goswami, Dibya Jyoti Hazarika, Pompi Das, Trishnamoni Gautom, Madhumita Barooah and Robin Chandra Boro*

Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat-13, India *Corresponding author

ABSTRACT

Studies on the and diversity of macrofungi have gained significance during the recent years, as many macrofungi are facing the risk of extinction because of habitat K e yw or ds destruction. We report, here a systematic and detailed study on the diversity and

distribution of wild in the state of Assam, India, based on phenotypic and Macrofungi, Diversity, Wild- molecular characteristics. A total 44 samples were collected from various locations of edible, Assam in different seasons during 2013-2015. Out of the total 44 samples, about 18 Morphological macrofungi occurred during summer season, 9 species were recorded during rainy, study, ITS. 13 species were frequently found during autumn, 5 species in winter and 8 species were prominent during spring. The soil was found as major habitat for 18 collected species, Article Info while 10 species were collected from the tree/hardwood tree. This study indicated that the

Accepted: diversity and distribution of macrofungi are dependent on the plant community and the 04 October 2017 environmental conditions. Molecular characterization based on rDNA -ITS- ((Internal Available Online: transcribed spacer) sequences revealed that the 44 samples belonged to 16 macrofungal 10 December 2017 families. Out of the 44 samples, 23 samples were reported to be edible and amongst the rest 21 non-edible strains, 5 strains have medicinal properties, 6 strains are poisonous, two has industrial application and the rest have not been fully explored.

Introduction

Fungi play a significant role in industry, Zygomycota) (Alexopoulus et al., 2000). agriculture, medicine, food industry, textiles, Macrofungi are found in varied types of and bioremediation (Danielson et al., 1989; habitats, depending on the composition of tree Manzi et al., 1999; Krzywinski et al., 2009; species and other substrates. The distribution Changet al., 2004). Fungi can be broadly of macrofungal species is low in hot and dry classified into three different forms viz., seasons while they are abundant in spring and yeast, moulds and macrofungi or mushrooms. autumn due to favourable climate and Macrofungi are large, conspicuous spore- abundance of flora during that time (Pilz et bearing structures and have fleshy, tough al., 2001). Similar to yeast and moulds, umbrella like sporophores that bear macrofungi also possess immense biological holobasidia on the surface of gills or lamellae and economic impact since they serve as that hang down from the cap belonging to food, medicine, biocontrol agents and produce basidiomycetes and ascomycetes (few under of bioactive compounds such as anti-oxidant, 275

Int.J.Curr.Microbiol.App.Sci (2017) 6(12): 275-297 anti-diabetic, hypocholesterolemic, anti- potential. Therefore, it is crucial to document tumor, anti-cancer, immunomodulatory, anti- the diversity, distribution and abundance of allergic, nephroprotective, and anti-microbial these macrofungi in Assam. Such study will agents (Patel et al., 2012). Macrofungi are not only pave the way towards domestication used in the bioremediation of industrial waste and commercialization of the wild species for and in the accumulation of heavy metals from economic benefits but will also help in the environment (Tuli et al., 2013). There are establishing a strong basis for molecular about 2166 worldwide recognized edible taxonomy. In this study we aimed to collect species that include about 470 species macrofungi from different habitats of Assam possessing medicinal properties. Furthermore, followed by their taxonomic evaluation using macrofungi are useful indicators of health or phenotypic and molecular characteristics. age of an ecosystem (Boa et al., 2004). Materials and Methods Assam, situated in the Northeast of India is a constituent unit of the Eastern Himalayan Collection of macrofungi Biodiversity Region; one of the two biodiversity “Hot Spots” in the country. The Systematic and periodical survey of different climatic conditions along with varied physical forests, tea gardens and other habitats were features in Assam, makes it rich in diversity undertaken during November, 2013 to April, of ecological habitats such as forests, 2015. For this study, the opportunistic grasslands, wetlands, which harbour and sampling of macrofungi protocol, was sustain wide range of floral and faunal followed (Mueller et al., 2004). The sampling species. Assam has a sub-tropical monsoon method involved walking throughout the climate with an average rainfall of around entire study region and collecting the 1,500 mm per year. The day-time temperature macrofungi covering up to 80% of the area. in summer, the rainy season, rises to around Sampling of the ground-dwelling macrofungi 35ºC and in winter cools to 25ºC with a night involved careful extraction of the fruit body time minimum of around 10ºC which is from the soil and leaf litter, ensuring that the favorsthe growth of most of the macrofungal basal surface was intact. The tree-dwelling species (Das et al., 2005). Although, macrofungi were sampled by cutting off the macrofungi are good resources for agro- basidiomata from the trees without disturbing industrial, medicinal (Veena et al., 2012) and the spore surfaces present below. Different commercial purposes, very few studies have species exhibit different fruiting phenologies, reported the macrofungal diversity of which vary from month to month and at Northeast India (Khaund and Joshi, different altitudes and regions. 2014). Several varieties of macrofungi are being consumed by the local tribes of Assam The survey and collection of was which are harvested from wild habitats but no done from different locations of Assam viz. efforts have been made to cultivate these Nalbari, Barpeta, Jorhat, Sibasagar, Karbi macrofungi for commercial production. Anglong, Kamrup, Golaghat, Goalpara, Moreover, there are no scientific reports Tezpur, Tinsukia, Rangia and Nagaon under related to characteristics of the wild edible different agro-climatic conditions, during the macrofungi which are slowly disappearing year 2013-2015. The macrofungal samples due to habitat loss. Because of these reasons, were collected in different seasons of the the farmers and entrepreneurs of Assam have year; rainy season (July and August), winter not been able to exploit its agro-economic (December and January), spring (March and

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April) and summer (May and June) during polymerase protocol (Takara, Japan) with 20 2013-15. Soft macrofungi were collected pmols of each primer, 2U Taq DNA carefully by using forceps/free hand, while polymerase and 50 ng genomic DNA in a the macrofungi growing on wood were final volume of 50 μL and amplification was collected along with small part of wood. All performed using a thermal cycler (Applied collected samples were carefully wrapped in Biosystems, USA). The PCR program was as aluminum foil, placed in an air-tight zip-lock follows: initial denaturing step of 94°C for 3 bag and labeled with the collection number, min, followed by 35 cycles of denaturation at location, date, and other data. The photograph 94°C for 30 sec, annealing at 49°C for 1 min of the mushroom was taken in their natural and elongation at 72°C for 1 min; a final habitat. extension step at 72°C for 7 min. The amplified products were sequenced as per the Morphological characterization BigDye Terminator sequencing protocol in an ABI 377 automated DNA Sequencer (Applied Morphological characterization of the Biosystems, USA). The ITS rDNA sequence macrofungal strains were carried out based on reads obtained after sequencing were the cap size, cap structure, colour of the assembled into contig using CodonCode fruiting body, dimensions of , gill shape Aligner (CodonCode Corporation, USA). and gill colour as described earlier (Kumar et Sequence similarity tool BLAST was al., 2015). Further, the fresh sporocarps of employed to find the similarity of the mushroom species were cultured to obtain sequences with known 16S rDNA sequences vegetative mycelium from the tissue (small in the GenBank database and the sequences pieces cut from section between the pileus were deposited in GenBank of NCBI to obtain and stipe) using Potato Dextrose Agar (PDA) the accession numbers. Phylogenetic analysis media and incubated at 25 ± 2°C. of the isolates was carried out based on ITS- rDNA sequences in MEGA6 software Molecular characterization and (Tamura et al., 2013). A phylogenetic tree phylogenetic analysis of the macrofungal was constructed using the ITS-rDNA strains sequences of the macrofungal strains along with other similar or related macrofungal ITS- For Molecular characterization of the 44 rDNA sequences retrieved from the NCBI macrofungal isolates were performed by GenBank. The sequences were aligned with sequencing the rDNA internal transcribed Clustal W using default parameters and a spacer (ITS) region. For DNA extraction, neighbor-joining tree was constructed using individual strains were cultured on PDA at 25 MEGA6 software (http://www.megasoftware. ± 2°C for 7 days to obtain mycelia. The net/) with Kimura-2 parameter correction and genomic DNA was extracted from the pure 1000-step bootstrap. mycelia using the protocol of Singh et al., (2003). Amplification of the ITS (Internal Results and Discussion transcribed spacer) region of the nuclear ribosomal repeat unit was done using Sample collection and morphological universal primer set ITS1 (5'- characterization TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') A total of 44 macrofungal samples were (Thatoi et al., 2014). PCR reaction mixture collected from different places of Assam, viz., was prepared as per TaKaRa Ex Taq® DNA Nalbari, Barpeta, Jorhat, Sibasagar,

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KarbiAnglong, Kamrup, Golaghat, Goalpara, al., (2016) documented wild edible Tezpur, Tinsukia, Rangia and Nagaon (Fig. 1) macrofungi from Shyrwat and Upper Shillong during the study period. The details of the Reserve Forests of Meghalaya, Northeast sample collection sites are given in Table 1. India where a total of 22 macrofungi were collected during the rainy season (July to Several workers have studied the diversity of September) 2014, and identified on the basis macrofungi from different parts of India of macroscopic and microscopic namely Kashmir (Sheikh et al., 2014), characteristics. The diversity of wild Garhwal (Vishwakarma et al., 2012), Tamil macrofungi in Nagaland, India was reported Nadu (Mani et al., 2009). However very less by Toshinungla et al., (2016) where a total of number of reports appeared from north 87 species of wild macrofungi were collected eastern part of India and only a few attempts and identified and out of the collected have been initiated to document macrofungi macrofungi, 37 species were identified as from this area. An attempt was made by edible, 21 species medicinal, 5 poisonous and Gogoi and Sarma to find out some edible 37 inedible/unclassified. Surveys were macrofungi of Dhemaji district of Assam conducted in Narpuh Reserve Forest, (Gogoi et al., 2012). Meghalaya, India to unveil the macrofungal diversity and identified a variety of The morphological taxonomic system of macrofungi on the basis of macro- and classification given by Ainsworth (1963) and microscopic characteristics. modified by Webster (2007) was used for characterization of the macrofungal samples Seasonal occurrence of the macrofungal collected from different locations (Table 2). species Macroscopic features of the carpophores pertaining to pileus, lamellae, stipe, etc. Climatic and edaphic conditions prevailing in which play an important role in the taxonomy Assam favoured the occurrence of diverse of macrofungi have been discussed. The macrofungi. Our study revealed that 18 photographs of the collected macrofungal species occurred during the summer season, 9 samples are shown in Figure 2(a) and 2(b). species were recorded during rainy, 13 The fruiting bodies of macrofungi were found species were frequently found during autumn, to be distinctly different from each other. 5 species during winter and 8 species grew Some species that grew on wood had caps mostly in the spring. Comparison of the that attach directly to the wood (e.g. seasonal occurrence is depicted in Figure 3. Pycnoporus, Ganoderma). In some cases, the Some species were observed to overlap two cap was semi-circular and attached by the seasons for e.g. sapidus, straight edge while in others the stalk (also Chlorophyllum molybdites were found both in called the stipe, or stem) may be central and rainy and autumn season; support the cap in the middle and in vitreus in both autumn and winter season; Pleurotus, the stalk was off-central or lateral. titubans and Auricularia sp. grew The morphological data of the collected both during summer and rainy season. samples are detailed in Table 2. Similar Moisture is one of the major factors studies were carried out by Ram et al., (2010) influencing the fruiting of macrofungi. During where several edible fleshy fungi were the rainy seasons, the availability of adequate collected from the wilds in Eastern Uttar moisture favor the growth of the macrofungi. Pradesh during the rainy season on dead and Species diversity was higher in the rainy decaying plant or animal remains. Kalita et seasons compared to the early dry seasons, as

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Int.J.Curr.Microbiol.App.Sci (2017) 6(12): 275-297 there is adequate moisture available during It was observed that the diversity of the the rainy seasons. While the macrofungal macrofungal species depend on the habitat species collected in the early dry season was (Fig. 4). The highest numbers of samples predominated by the , since there were from Golaghat followed by Nagaon and is a decrease in rainfall and relative humidity Goalpara districts of Assam. The soil was along with increase in temperature and found to be the best source of diverse sunshine, most of the fleshy macrofungi macrofungal species followed by tree and cannot withstand these conditions. deadwood. A variety of habitats i.e. soil, tree, wood, litter, lawn, etc. supports growth for Singh and Prasad (2003) recorded a number macrofungi and soil was found as major of mushroom spp. during 1997-98 and 1998- habitat for 18 collected species, while 10 1999. The occurrence of species was found to species were collected from the be greater in September during 1997-98 and tree/hardwood tree. The variation observed in in August in 1998-99, while July and October occurrence of mushroom species in various exhibited a lesser number of macrofungi. habitats may be due to their particular mode However, in another investigation, July month of nutrition; the macrofungi growing on the of each year (2002-05) showed a greater soil are symbiotic, on rotting and dead wood number of macrofungal species. A varying are saprophytic and on trees are parasitic. number of macrofungi of different species in Cosmopolitan distribution of macrofungi have different months may be due to low relative also been reported (Suryanarayanan et al., humidity, low temperature range, very little 2004), certain species of mushroom are and no rainfall. associated with particular kinds of trees and plants (Hawksworth, 2001).

Table.1 GPS data for the macrofungal sample collection sites

Sites Latitude Longitude Elevation(m) Mariani 26°39′26″ N 94°18′55″ E 118 Goalpara 26° 10' 12.8028'' N 90° 37' 35.8212'' E 44 Jorhat 26° 45' 27N 94° 12' 11E 111 Diphu 25°50′36″N 93°25′52″E 2000 KarbiAnglong 26º35' N 93º50' E 186 Sibsagar 26° 59' 3N 94° 38' 16E 95 Kamrup 26° 26' 57N 91° 36' 49E 38 Golaghat 26° 30' 42N 93° 57' 34E 90 Barpeta 26° 19' 22N 91° 0' 23E 27 Tezpur 26° 37' 60N 92° 47' 60E 47 Nagaon 26° 21' 0N 92° 40' 0E 54 Tinsukia 27°29'30.63"N 95°20'55.30"E 125 Rangia 26°26'28.61"N 91°37'7.11"E 53

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Table.2 Morphological data of the collected macrofungal samples

Sl. Samples Site of Habitat Fruiting Stipe Pileus/Gills No Collection Body/Colour 1. M3 Mariani Tree Sessile, corky. Absent. A small, 5 cm in length. Scarlet red to single Pores on the flame orange attachment point underside 2. Assam Mariani Hardwood Flesh is white, Absent. Cap Fan or oyster- tree firm, and varies in laterally attached shaped (11 cm). thickness. White Decurrent with to grey or tan to white to cream dark-brown colour gills 3. T1 Goalpara Hardwood Woody brackets. Absent Fan-like or hoof- tree Cream to dark- like form brown honeycomb gills 4. O Titabor, Rice straw Flesh is white, Absent. Cap Fan or oyster- Jorhat firm, and varies in laterally attached shaped thickness 5. Sap Diphu Rice straw Flesh is white, Absent. Cap Fan or oyster- firm, and varies in laterally attached shaped (9 cm). thickness Decurrent 6. CN AAU, Jorhat Soil Flesh is white, Absent Conical (8.5 cm). spongy, and varies White to cream in in thickness colour 7. H10 AAU, Soil Fleshy and varies Present (7cm) Depressed (4 cm). Jorhat in Extended outwards thickness. Brown (brown in colour) 8. K Karbi Dead wood Tough with a Tapering to base Depressed (7 cm). Anglong well-developed and a persistent Lamellate, (white central stalk. Flesh ring is present on to cream in colour) fibrous. White to stipe (4 cm x pale brown 7mm) 9. S1 Goalpara Tree Flesh is white, Tapering to base. Fan or oyster- firm, and varies in shaped (6 cm) thickness Decurrent white to cream in colour 10. AM Sibsagar Soil Flesh is cream, A persistent ring Flat scales (4.3 cm) and varies in is present on serrated, white to thickness. Grey to stipe (7cm x 20 cream in colour pale brown mm) 11. HN Kamrup Hardwood Corky, hard, Absent Fan-like or hoof- tree woody- like form. textured. dark- Decurrent (grey to brown brown in colour) 12. N1 Diphu Soil Brown spongy Absent Radicating, fleshy well-developed (4.6 cm) central stalk. Flesh fibrous. 13. V AAU, Jorhat Hardwood Corky, hard, Absent Kidney shaped (12 tree woody- cm). Decurrent textured. dark- (brown in colour) brown

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14. N2 Rangia Soil White with shaggy A loose ring Gills change scales around the stipe rapidly from white of 8 cm length to pink, then to black. 15. P2 Golaghat Soil Short brown blunt Radicating, Depressed (1.3 elevated lines on fleshy (4.6cm) cm). Decurrent the lower surface (brown in colour) of the cap and well developed with central stalk 16. T2 Barpeta Tree Blunt elevated Absent Depressed (4.3 lines on the lower cm).Free (cream in surface of the cap colour) without central stalk 17. H1 Tezpur Tree, Wood Shiny, dry and 9 cm x 6 mm, 6 cm wide, scaly extremely with a slight ring lamellate gills on tough with a well- near the apex). lower surface, developed central decurrent, edge stalk. Whitish to sterile, denticulate buff with cinnamon-brown scales. 18. C1 AAU, Jorhat Soil Flesh is cream in Absent 3 cm wide, top- colour, and varies shaped. white to in thickness. cream in colour whitish -brown 19. G2 Nagaon Soil Elevated lines on 4 cm x 3 mm, Umbonate. Free the lower surface with a slight ring (cream in colour) of the cap without near the apex) central stalk. Yellowish white 20. H2 Golaghat Soil Blunt elevated Fleshy (5.6cm) Depressed (1.3 cm) lines on the lower brown in colour surface of the cap with central stalk. whitish 21. H4 Jorhat Soil Blunt elevated Radicating, Umbonate. Free lines on the lower fleshy (2.3cm) (cream in colour) surface of the cap without central stalk 22. H7 Golaghat Soil Blunt elevated Tapering to base Flat (9.3 cm). lines on the lower and a persistent (Greyish in colour) surface of the cap ring with central stalk 23. H6 Nagaon Soil Brownish elevated Tapering to base Umbonate. Free lines on the lower and a persistent (cream in colour) surface of the cap ring is present on without central stipe stalk 24. AP2 Barpeta Soil a brick-red Light yellow Crowded, colouration in the (4.5–7 cm in yellowish center diameter)

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25. AP3 Golaghat Dead tree Basidiocarps, 5 5 mm thick, Pore surface white mm thick, waxy greyish pore to bluish white and and soft when surface translucent fresh, hard and cartilaginous when dry 26. AP5 Barpeta rotting wood Cream or light Centrally White; up to 3.5 covered in a fine depressed cm long and 2.5 cm white bloom attached to the diameter convex, 4-12cm substrate via an eccentric stem 27. AP6 Sibsagar Wood Oyster shell- Absent Decurrent, running shaped to fan- a short ways down shaped and colour the stipe–10 mm ranges from ivory broad, white to white to pinkish cream in colour buff to orange- grey 28. AP7 Sibsagar Soil Pale fawn tan or Stem 13 cm x 3- Pileus 1.5-8 cm pale brownish 10 mm, centric wide, densely towards the centre to excentric decurrent gills 29. AP8 Tinsukia Soil Brownish or Pale grey or The underside greyish cap brown stem (8 bears soft, pale measures 17 cm in cm) and (3.1 in) grey „teeth‟ rather diameter and is high and 3 cm than gills covered with (1.2 in) wide coarse darker brown scale 30. AP9 Nagaon Wood Fibrous-corky, Absent Soft tomentose, usually 3–10 cm semi-circular to in elongate Pore diameter,attached surface pale orange directly to the substrate 31. AP11 Tinsukia Bark of Tree Gelatinous, Absent Irregular shape, orange-yellow and usually breaks fruit body of the through the bark of , which can dead branches grow up to 4.5 cm diameter 32. AP12 Tinsukia Lawn White or pale 7 cm tall, the White and finely yellowish parallel stem scaly, the cap developing with a small pressed-down double ringed fibers or scales bulb at its base 33. AP13 Goalpara Tree Basidiocarps Absent Cap surface is pale consist of tan to dull chestnut numerous rosette- brown with finely like flattened fan- fibrillose tiny shaped pileus scales (squamules) 34. AP14 Goalpara Soil Cap is 1.5 - 5 cm, 3-10 cm tall and Free from the stem and grows from 2-4 mm wide, and fragile. egg-shaped when whitish-yellow Whitish or pale young to broadly colour yellowish to rusty

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convex cinnamon 35. AP15 Golaghat Forest Smaller yellow-to- White or pale Pink and crowded, lilac-capped pink, smooth the broad gills are bonnet mushroom with longitudinal deeply sinuate fibres, with no stem ring 36. AP16 Golaghat Dead wood White, thin and Central or Running down the tough scaly with slightly off- stem; whitish brown to golden center; 2.5 cm brown scales and long; 3 mm fibrils wide; brown to yellowish brown; scaly to hairy 37. AP17 Dergaon Tree Colour of the cap 3 cm long; 1 cm Running down the is purple to wide; tough; off- stem; crowded; pinkish brown centre densely purplish. which is densely hairy Gills decurrent haired 38. AP1 Titabar Dead log Yellow to brown The stalk is thick Gills decurrent and has scales and short, 5 cm long 39. AP19 Kamrup Soil Orange in colour Overlapping Wide, shaped like a and has a calfskin- clumps, and 4 fan and attached like texture cm thick direct to the trunk of a tree 40. AP20 Kamrup Soil Brown 5 cm tall, 2.5 Gills adnexed, hygrophanous cap mm thick, moderately broad (striate when slender, thin, fresh), the disc fragile usually dingy yellowish-brown 41. AP21 Jorhat Litter Yellow to brown 8 cm long and 2 Adnate; crowded; with orange caps cm in diameter, yellow cylindrical; smooth, with fine longitudinal fibres 42. AP2 Golaghat Tree Flesh is white, 14 cm, convex, Gills: Decurrent firm, and varies in Short and offset thickness. White from the centre to grey or tan to of the cap dark-brown 43. AP24 Dergaon Dead log Reminiscent of a Attached to the Gelatinous gills floppy ear, cup- substrate shaped and has a laterally with tough, gelatinous, very short stalk elastic texture 44. AP25 Nagaon Lawn Brown or beige to 6 cm tall x 2 mm Gills are adnate or straw colour thick, equal, adnexed, grey to hollow, straw purple-brown with colour and whitish annulus absent

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Table.3 Nucleotide nblast results of the isolates

Sl.No Samples Sequencing Result Accession No. 1. M3 Pycnoporus coccineus KJ862075 2. Assam Pleurotus sapidus KJ862064 3. T1 Ganoderma lucidum KJ862063 4. O Pleurotus sapidus KJ862076 5. Sap KM609393 6. CN Lycoperdon pyriforme KM609394 7. H10 Lactarius friabilis KM609395 8. K Lentinus sajorcaju KJ862073 9. S1 Pleurotus floridanus KJ862077 10. AM Chlorophyllum molybdites KM609396 11. HN Ganoderma sp KM609397 12. N1 Lentinus sp KM609398 13. V Ganoderma applanatum KM609399 14. N2 Coprinus spp. KM609400 15. P2 Agrocybe pediades KM609401 16. T2 Ganoderma sp KM609402 17. H1 Lentinus critinus KM609403 18. C1 Hymenopellis megalospora KM609404 19. G2 Leucoagaricus leucothites KM609405 20. H2 campestris KM609406 21. H4 Macrolepiota rhacodes KM609407 22. H7 Macrolepiota procera KM609408 23. H6 Coprinus disseminatus KM609409 24. AP2 Hypholomas ublateritium KR824074 25. AP3 KR824075 26. AP5 Pleurotus cornucopiae KR824077 27. AP6 Pleurotus populinus KR824078 28. AP7 Panus lecomtei KR824079 29. AP8 Sarcodon imbricatus KR824080 30. AP9 Fomitopsiso streiformis KR824082 31. AP11 Dacrymyces sp KR824083 32. AP12 Agaricus arvensis KR824084 33. AP13 giganteus KR824085 34. AP14 Bolbitius titubans KR824086 35. AP15 rosea KR824087 36. AP16 Polyporus arcularius KR824088 37. AP17 Lentinus strigosus KR824089 38. AP1 Polyporus squamosus KR824090 39. AP19 Laetiporus sulphurous KR824091 40. AP20 corrugis KR824092 41. AP21 Gymnopilus dilepis KR824093 42. AP2 KR824093 43. AP24 Auricularia sp KR824096 44. AP25 Psilocybe mexicana KR824097

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Table.4 Classification study of macrofungi from different locations of Assam

ORDER FAMILY SPECIES USE Pycnoporus coccineus Industrial Lentinus sajorcaju Edible Lentinus sp. Edible Polyporaceae Lentinus critinus Edible Lentinus strigosus Edible Laetiporus sulphurous Edible Panus lecomtei Edible Polyporus squamosus Medicinal Polyporus arcularius Edible Physisporinus vitreus Industrial Edible Polyporales Fomitopsidaceae Fomitopsis ostreiformis Non-Edible

Ganoderma sp. Non-Edible Ganoderma applanatum Medicinal Ganodermataceae Ganoderma lucidum Medicinal Ganoderma sp. Non-Edible Pleurotus Sapidus Edible Pleurotus Sapidus Edible Pleurotus ostreatus Edible Pleurotus floridanus Edible Pleurotus cornucopiae Edible Pleurotus pulmonarius Edible Pleurotus populinus Edible Agaricaceae Lycoperdon pyriforme Edible Coprinus sp. Non-Edible Agaricus arvensis Edible Macrolepiota rhacodes Non-Edible*

Macrolepiota procera Non-Edible*

Chlorophyllum molybdites Non-Edible* Coprinus disseminatus Non-Edible* Leucoagaricus leucothites Non-Edible Agaricus arvensis Edible Agaricus campestris Edible Bolbitius titubans Non-Edible Mycena roseae Non-Edible Strophariaceae Agrocybe pediades Non-Edible* Psilocybe mexicana Non-Edible* Hypholoma sublaterium Non-edible Physalacriaceae Hymenopellis megalospora Non-Edible Cortinariaceae Gymnopilus dilepis Non-Edible Psathyrella corrugis Non-Edible Russulales Russulaceae Lactarius friabilis Non-Edible Thelephorales Bankeraceae Sarcodon imbricatus Edible Dacrymycetales Dacrymycetaceae Dacrymyces sp. Non-Edible Auriculariales Auriculariaceae Auricularia sp. Medicinal *Poisonous macrofungi (Thatoiet al., 2004) 285

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Fig.1 a) Map of India highlighting Assam b) Satellite image of Assam with different districts of Assam c) Satellite image of Assam showing different sites of collection of macrofungi

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Fig.2 (a) Fruiting bodies of the collected samples. 1= M3, 2=Assam, 3 =T1, 4=O, 5= Sap, 6= CN,7=H10, 8= K, 9=S1,10= AM,11= HN,12= N1,13= V, 14= N2,15= P2,16= T2,17= H1,18= C1,19= G2, 20=H2, 21=H4, 22= H7, 23= H6, 24=AP2

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Fig.2 (b) Fruiting bodies of the collected samples. 25= AP3, 26= AP5, 27= AP6, 28= AP7, 29= AP8, 30= AP9, 31= AP11, 32= AP12, 33= AP13, 34=AP14, 35= AP15, 36= AP16, 37= AP17, 38= AP1, 39= AP19, 40= AP20, 41= AP21, 42= AP2 43= AP24, 44=AP25)

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Fig.3 Seasonal distribution of the macrofungal species. Rainy season (July and August), winter (December and January), spring (March and April) and summer (May and June) during 2013-15

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January February Ma April May June July August September October November December Pycnoporus coccineus Pleurotus sapidus Ganoderma lucidum Pleurotus sapidus Pleurotus ostreatus Lycoperdon pyriforme Lactarius friabilis Lentinus sajorcaju Pleurotus floridanus Chlorophyllum molybdites Ganoderma sp Lentinus sp Ganoderma applanatum Coprinus spp. Agrocybe pediades Ganoderma sp Lentinus critinus Hymenopellis megalospora Leucoagaricus leucothites Agaricus campestris Macrolepiota rhacodes Macrolepiota procera Coprinus disseminatus Hypholomas ublateritium Physisporinus vitreus Pleurotus cornucopiae Pleurotus populinus Panus lecomtei Sarcodon imbricatus Fomitopsiso streiformis Dacrymyces sp Agaricus arvensis Meripilus giganteus Bolbitius titubans Mycena rosea Polyporus arcularius Lentinus strigosus Polyporus squamosus Laetiporussulphurous Psathyrella corrugis Gymnopilus dilepis Pleurotus pulmonarius Auriculariasp Psilocybe mexicana

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Fig.4 CIRCOS illustration of the samples depicting the relationship between the v macrofungi with that of the site of collection and habitat

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Fig.5 Evolutionary relationships of macrofungal species (NJ Tree Method)

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Fig.6 Scientific classification of the identified strains based on molecular data

Molecular characterization of the 4.1 (CodonCode Corporation, USA). The macrofungal strains consensus sequence obtained for each strain was compared with the ITS rDNA sequences The genomic DNA was subjected to PCR- of fungi available in NCBI through a BLAST amplification for the ITS region of the rDNA search and the sequence data has been using ITS1 and ITS4 primers. ITS regions of submitted to GenBank, NCBI and the fungal ribosomal RNA are highly variable accession numbers were obtained (Table 3). sequences, which can be useful markers for The sequences were aligned with the help of determining fungal species. The approximate Codoncode Aligner and analysed for size of the amplified PCR products (~750bp) homology using nBLAST (NCBI) to obtain was purified and sequenced using ABI 377 possible identities (Altschul et al., 1997). The automated DNA Sequencer (Applied BLAST results displayed 97-99% homology Biosystems, USA) and resulting contigs were with the 44 isolates under study (Table 3). analyzed with CodonCode Aligner version The comparison of ITS gene from different

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Int.J.Curr.Microbiol.App.Sci (2017) 6(12): 275-297 strain and different species indicates that there East Hill region of India and confirmed exists high species-specific homogeneity. ninety-five species of higher fungi, among these, 85 species were new records. Recently, Many molecular markers have been applied in diversity of wild edible macrofungi from phylogenetic studies in order to decipher the Khasi hills of Meghalaya, India have been relationships among species of reported and a total of 11 different species Basidiomycetes (Shtaer et al., 2005; Shnyreva were identified based on spore morphology and Shtaer, 2006). However, internal (Khaund and Joshi, 2013). Three new species transcribed spacer (ITS) I and II the two non- of Russula (Russulasharmae, R. dubdiana and coding polymorphic regions, located between R. sikkimensis) from Sikkim (India) have been highly conserved regions of 18S, 5.8S and reported by Das et al., (2013). 28S rRNA genes (Agarwal et al., 2015), have been widely employed to assess the Phylogenetic analysis phylogenetic relationships of mushroom species (Choi et al., 2007). In India, Singh et Phylogenetic analysis was carried out by al., (2006) first reported molecular comparing the query sequences against a non- characterization of mushroom using ITS redundant database for comparative analysis primers specific to macrofungi and identified of ITS partial sequences. The phylogenetic 18 mushroom germplasms based on ITS tree generated through the Neighbor-Joining sequence polymorphism. Seven of these (NJ) method was well supported by the isolates were identified as Podaxis pistillaris, bootstrap values within their nodes. The six belonged to Phellorinia herculean, four phylogenetic analysis using partial ITS rRNA were identified as belonging to Phellinus gene sequences revealed that the isolate igniarius and only one was identified as showed closeness with each other. However, Gymnopilus subearlei. A recent report by further such studies involving more number Rajaratnam et al., (2012) indicated successful of isolates (taxa) can better depict their identification of wild macrofungi based on phylogenetic position at a community level. ITS (ITS1 and ITS4) sequence. Their study Phylogenetic analysis using the Neighbour- revealed that the wild mushroom belonged to Joining tree of the identified strains is shown which was a new report of a in Figure 5. The evolutionary history was mushroom in India. Some of the wild edible inferred using the Neighbor-Joining method macrofungi have also been reported from and the percentage of replicate trees in which Manipur and Arunachal Pradesh of North the associated taxa clustered together in the East India (Sing et al., 2002). Few strains of bootstrap test (1000 replicates) are shown Basidiomycetes have also been reported from next to the branches (Efronet al., 1996). The Assam (Baruah et al., 1971). tree is drawn to scale, with branch lengths in the same units as those of the evolutionary Verma et al., (1987) described fleshy fungal distances used to infer the phylogenetic tree. flora from Manipur and Meghalaya belonging All the 44 macrofungal isolates were grouped to the family Auriculariaceae, Clavariaceae, into ten clusters (Fig. 5), Cluster I comprised Cantharellaceae, Tricholomataceae, of 7 macrofungal isolates (Macrolepiota Pluteaceae, Paxillaceae, Cortinariaceae, rhacodes, Macrolepiota procera, Lycoperdon Cycoperdaceae, and Sclerodermataceaeof pyriforme, Leucoagaricus leucothites, Basidiomycotina and Halvellaceae of Psilocybe Mexicana, Agaricus arvensis, Ascomycotina. Again, Verma et al., (1995) Agaricus campestris), Cluster II comprised of carried out a macrofungal survey of the North 6 macrofungal isolates (Hypholomas

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Int.J.Curr.Microbiol.App.Sci (2017) 6(12): 275-297 ublateritium, Bolbitius titubans, Lactarius sp., It was observed that, among the 44 samples, Chlorophyllum molybdites, Panuslacomtei, 23 strains were edible and among the 21 non- meripiliusgigantius), Cluster III comprised of edible strains, 4 strains have medicinal only 1 macrofungal isolate (Fomitopsiso properties, 6 strains are poisonous, one has streiformis), Cluster IV comprised of 3 industrial application and the rest are not fully macrofungal isolates (Pycnoporus coccineus, explored. Singer (1986) recognized 230 Polyporus arcularius, Polyporus squamosus), genera and 5658 species spread over 17 Cluster V comprised of 5 macrofungal families in order Agaricales. Previous reports isolates (Ganoderma applanatum, worldwide indicated the edibility and Ganoderma lucidum, Ganoderma sp., medicinal properties of these species and a Laetiporus sulphurous, Ganoderma sp.) complete list of all macrofungal species and Cluster VI comprised of 2 macrofungal countries can be retrieved from isolates (Hymenopellis megalospora, Mycena www.wildusefulfungi.org (Schwarze et al., rosea), Cluster VII comprised of 4 2011). macrofungal isolates (Dacrymyces sp., Gymnopilus dilepis, Agrocybe pediades, Scientific classification of the identified Auriculariasp.), Cluster VIII comprised of 3 strains based on their molecular data has been macrofungal isolates (Coprinus disseminates, depicted in Figure 6. The Psythrallacorrugis, Coprinus sp.), Cluster IX contains about 30,000 described species, comprised of 2 macrofungal isolates which is 37% of the described species of true (Physisporinuscitreus, Sarcodon imbricatus) fungi (Kirk et al., 2001). Zhao et al., (2011) and Cluster X comprised of 11 macrofungal provide an overview of recent work on isolates (Pleurotus populinus, Pleurotus understanding phylogenetic relationships floridanus, Pleurotus sapidus, Pleurotus within the Agaricales, a major group within ostreatus, Pleurotus sapidus, pulmonarius, the Agaricomycotina that is the largest clade Pleurotuscornucupaie, Lentinus sp., Lentinus of mushroom-forming fungi (Matheny et al., critinus, Lentinus strigosus, Lentinus 2006). sajorcaju). A timely research regarding isolation, Classification of the macrofungal species identification and characterization of the existing mushroom flora is essential due to Classification of the macrofungal species over exploitation and habitat destruction. (Table 4) revealed that all the 44 strains Thus, our study was an attempt to survey; belonged to the division Basidiomycota. The collect valuable wild macrofungi and their macrofungal species can be distributed into identification based on phenotypic and 16 families viz. Polyporaceae and molecular characteristics, to know the Agaricaceae (10 species each), Pleurotaceae mycotreasure available in this region that is (6 species), Ganodermataceae (4 species), yet to be fully explored. In this study we Meripilaceae and Strophariaceae (2 species observed that 23 strains were edible and the each), Fomitopsidaceae, Russulaceae, rest 21 were non-edible. Among the non- Bolbitiaceae, Mycenaceae, Physalacriaceae, edible strains, 4 strains have medicinal Cortinariaceae, Psathyrellaceae, Bankeraceae, properties, 6 strains are poisonous, one has Dacrymycetaceae and Auriculariaceae (1 industrial application and the rest are not fully species). The order Agaricales consisting of explored. Further studies on this less explored 23 species was found to be dominant followed may provide valuable information as by Polyporales with 17 species in this region. macrofungi have a great potential for the

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How to cite this article:

Assma Parveen, Lipika Khataniar, Gunajit Goswami, Dibya Jyoti Hazarika, Pompi Das, Trishnamoni Gautom, Madhumita Barooah and Robin Chandra Boro. 2017. A Study on the Diversity and Habitat Specificity of Macrofungi of Assam, India. Int.J.Curr.Microbiol.App.Sci. 6(12): 275-297. doi: https://doi.org/10.20546/ijcmas.2017.612.034

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